Lymphocytes are a subset of leukocytes (white blood cells). Within the lymphocyte population, at least two distinct cell types have been identified that are able to lyse other cells, including autologous malignant cells, autologous virus-infected cells, and allogeneic cells. These two predominant cytotoxic lymphocytes are cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.
All T lymphocytes, including CTL, express on the plasma membrane and/or in the cytoplasm an antigen referred to as CD3, along with numerous other antigens that are associated with various T-cell subtypes. CD3 is composed of at least three proteins of 20-30 kD that are non-covalently associated with the T-cell-antigen receptor (i.e., the structure responsible for antigen recognition). Numerous antibodies have been produced against CD3, including the anti-Leu-4 monoclonal antibody (commercially available from Becton Dickinson Monoclonal Center, Inc.). Most CTL lyse target cells (for example, virus-infected target cells) provided that the target cells express syngeneic major histocompatability (MHC) antigens. Accordingly, such cells are subject to MHC restriction.
NK cells differ from T lymphocytes in that most NK cells do not express either CD3 antigen or the T-cell-antigen receptor (see Lanier et al., J. Immunol. (1986) 137:2735). NK cells are also able to lyse certain tumor cells and virus-infected cells, but are not MHC restricted.
Whereas T cells can be readily identified by their reactivity with anti-CD3 antibodies, there has been no simple and sensitive method for the identification or enumeration of NK cells using a single labeling reagent. It is well known that most NK cells express the CD16 antigen. CD16 is a 50-70 kD glycoprotein that is associated with a receptor for IgG. Numerous antibodies have been produced against CD16, including anti-Leu-11a, VEP13, B73.1, L23 and others (Lanier et al., J. Immunol. (1983) 131:1789; Perussia et al., J. Immunol. (1983) 130:2133; Perussia et al., J. Immunol. (1983) 130:2142; Rumpold et al., J. Immunol. (1982) 129:1458; Lanier et al., J. Immunol. (1986) 136:4480). However, it has been demonstrated that CD16 cannot be detected on all NK cells, particularly NK cells that have been activated in culture. Moreover, in certain circumstances CD16 can also be expressed on T lymphocytes (Lanier et al., J. Exp. Med. (1985) 162:2089). Most NK cells express another glycoprotein on the plasma membrane that is identified by the anti-Leu-19 monoclonal antibody, an antibody commercially available from the Becton Dickinson Monoclonal Center, Inc. Anti-Leu-19 recognizes a glycoprotein of about 160 kD (GP160) that is also recognized by the NKH-1 monoclonal antibody (Lanier et al., J. Immunol. (1986) 136:4480). However, neither anti-Leu-19 nor NKH-1 react exclusively with NK cells but can also react with other non-lymphocyte cell types (Lanier et al., J. Immunol. (1987) 138:2019). Anti-Leu-19 also reacts with a unique minor subset of T lymphocytes, at least some of which kill without MHC restriction (Lanier et al., J. Immunol. (1986) 136:4480). Finally, the amount of the antigen recognized by anti-Leu-19 on the plasma membrane of NK cells is often low, making it difficult to precisely identify and enumerate the number of NK cells in a mixed cell population, such as blood or other tissues.
Recently, a new method of cancer therapy has been developed that involves the combined use of in vivo interleukin-2 (IL-2) drug treatment and administrating the patient's autologous lymphocytes that have been activated in vitro with IL-2, often referred to as lymphokine-activated killer (LAK) cell and IL-2 therapy (Rosenberg et al., N. Eng. J. Med. (1985) 313:1485). There is evidence that the cytotoxic effector cells are predominantly NK cells (Phillips and Lanier, J. Exp. Med. (1986) 164:814). Thus, there is a clear need for a method to identify, enumerate, and monitor the levels of NK cells and T lymphocytes in this type of therapy, as well as in other clinical situations to evaluate activation or suppression of the immune system.
Prior to the present invention unequivocal simultaneous identification of NK cells and T lymphocytes was not possible using a single assay. Accordingly, there remains a need for distinguishing between NK cells and T cells using techniques that can be simpilifed and, in preferred embodiments, automated.